Dbl proteins act as guanine nucleotide exchange factors (GEFs) for Rho family GTPases by enhancing GDP dissociation and facilitating GTP binding in vivo, thereby promoting biological activation. Emerging evidence implicates Rho proteins in many signaling pathways leading to regulation of cell shape, cell attachment, cell motility and invasion, cell-cell interactions, cell proliferation, differentiation, and apoptosis. Up-regulation of Dbl function is believed to populate Rho GTPases in their biologically active GTP-bound form, thereby leading to increased cell transformation and tumorigenesis. The overall goal of our proposal is to characterize the mode of binding and binding specificity of Rho GTPase substrates for the Dbl proteins, Dbs and Vav2, and investigate the mechanism of guanine nucleotide exchange. A series of multi-disciplinary studies aimed at elucidating the relationship between GEF catalytic function in vitro and the cell growth regulatory roles of these Dbl proteins in vivo are proposed. These efforts include biophysical and structural characterization, biochemical analyses, and molecular and cell biology approaches. Our research efforts will be focused on two Dbl family members, namely Dbs and Vav2. Both of these Dbl proteins are amenable to detailed structural and biochemical analyses in vitro, act as multi-functional exchange factors for Rho GTPases, and possess defined biological properties that can be assessed in vivo. The research efforts proposed herein should not only provide critical insight in the regulation and signaling properties associated with Dbs and Vav2, but given the conserved nature of Dbl homology (DH) and Pleckstrin-homology (PH) domains contained in Dbl proteins, should also aid in understanding the regulation of other Dbl family members. The specific aims of this proposal are outlined below: Specific Aim 1 - Elucidate the role of Dbl homology (DH), Pleckstrin-homology (PH), and cysteine-rich (CRD) sub-domains of Vav2 and Dbs in determining binding specificity and guanine nucleotide exchange activities toward Rho GTPase substrates in vitro. Specific Aim 2 - Identify and characterize mutations in the DH, PH, and CRD domains of Vav2 and Dbs that will enable identification of residues and generation of reagents useful for assessing Rho GTPase binding, specificity, catalytic function and interactions with inositol phospholipids. Specific Aim 3 - Delineate the roles of the DH, PH, and CRD domains of Vav2 and Dbs in promoting guanine nucleotide exchange and Rho GTPase-mediated cell signaling and transformation in vivo. Specific Aim 4 - Investigate the structural and dynamic features of DH, PH and CRD domains, contained within Dbs and Vav2, in the presence and absence of Rho GTPase substrates and inositol phospholipids